1. Introduction
The keeping of South American camelids (SACs)—especially alpacas (
Vicugna pacos) and llamas (
Lama glama)—is becoming more and more popular in Europe [
1,
2,
3]. Due to their natural environment in the Andes, SACs are adapted to high exposure to ultraviolet (UV) radiation, which is important for vitamin D
3 synthesis in the skin [
4]. As vitamin D
3 is a precursor of 1,25-dihydroxycholecalciferol, a hormone involved in calcium and phosphate homeostasis, deficiencies in vitamin D
3 can lead to disorders in bone metabolism [
5]. As UV radiation is lower in Central Europe compared to the natural habitat of SACs in the Andes, vitamin D
3 deficiency is often diagnosed in SACs in Central and Northern Europe. Plasma vitamin D
3 levels depend, among others, on pigmentation of the animals and the season [
6]. Particularly in fall and winter, there is a decrease in vitamin D
3 plasma levels in SACs [
7,
8]. Vitamin D
3 deficiency is known to be responsible for low plasma calcium and phosphate levels and the development of rickets in growing camelids [
6,
9,
10]. Being aware of this fact, many owners supplement their SACs, especially crias, with vitamin D
3, either by feed additives or parenterally. Different vitamin D
3 sources are also often combined. Various recommended dosages for vitamin D
3 supplementation of SACs have been published by different authors [
11,
12,
13]. This increases the risk of an overdosage by high vitamin D
3 concentrations in ad libitum mineral feed or by parenteral injection. In the case of the animals in the present study, the attending veterinarian followed the recommendations of the scientific literature. Nonetheless, in the corresponding chapter of the textbook, the units (1 µg corresponds to 40 I.U.) were mixed up [
14], which led to an overdosage of vitamin D
3 by a factor of 40.
High plasma vitamin D
3 levels were reported to induce calcinosis with calcifications of different organs in several domestic and wild mammals [
15,
16,
17,
18]. Toxic effects of excessive administration of vitamin D are thought to occur because of increased concentrations of 25-hydroxycholecalciferol; there is a high capacity of the liver for hydroxylation at position 25, while the hydroxylation at position 1, resulting in the production of 1,25-dihydroxycholecalciferol, is tightly regulated by parathyroid hormone and calcium. Pharmacological plasma concentrations of 25-hydroxycholecalciferol result in an activation of the vitamin D receptor, thus exerting effects similar to 1,25-dihydroxycholecalciferol. It has been shown, in k.o. mice lacking the enzyme that is pivotal for the hydroxylation at position 1, that 25-hydroxycholecalciferol can bind to the vitamin D receptor, although with lower affinity in comparison to 1,25-dihydroxycholecalciferol. In addition, high concentrations of vitamin D metabolites could result in more competition for the vitamin D binding protein, consequently, more free and therefore biologically active 1,25-dihydroxycholecalciferol, although total concentrations remain unaltered. The activation of the vitamin D receptor, either by 1,25-dihydroxycholecalciferol or by 25-hydroxycholecalciferol, increases not only plasma concentrations of calcium, but also of phosphate [
19,
20].
Hypercalcemia and hyperphosphatemia lead to calcification of various tissues. The first affected organs are the heart, the circulatory system and the kidneys [
18,
21]. Cases of calcinosis in alpacas causing damage to the arteries or the kidneys have been previously reported [
22,
23,
24,
25]. However, there is a lack of available data concerning the impact of vitamin D
3 intoxication on the functional aspects of kidney parameters in SACs.
Therefore, the present study investigates severe cases of iatrogenic vitamin D3 intoxication in three alpaca crias, with a special focus on changes in kidney function.
4. Discussion
The presented cases show a severe impact of vitamin D
3 intoxication on the renal function in alpaca crias, which led to death after an acute kidney failure. Similar vitamin D intoxications have been reported in Airedale puppies, after an oral intake of vitamin D
3 of 200,000–250,000 IU/kg body weight over several days [
16], and also in cats, after being fed a commercial cat feed with overdosed vitamin D [
37]. In human medicine, there are also reports of vitamin D intoxication after manufacturing and labeling errors of dietary supplements [
38,
39]. In the presented case, overdosage was the result of a mixing up of the vitamin D units (I.U. versus mg/mL) in a veterinary textbook the local vet had used as reference, which resulted in a 40 times overdosage of vitamin D
3.
An oral overdosage of vitamin D in calves was reported to lead to Hyena disease with early calcification of epiphyseal plates [
15,
40]. Kidney failure due to vitamin D
3-induced calcinosis in alpaca crias has been previously reported in North America by Gerspach et al. [
22] and Jankovsky et al. [
25]. In these previous cases reported by Gerspach et al., the affected crias had been supplemented with vitamin D orally, either by colostral supplements or a paste, up to a total cumulative dose of 231,000 and 500,000 IU vitamin D
3. Calculated with the given 8.8 kg and 7.7 kg body weight that dose amounted to about 26,250 and 64,900 IU vitamin D/kg, respectively [
22]. The cria in the report by Jankovsky et al. had been treated with 100,000 IU vitamin D at a body weight of 9 kg, which corresponds to a dose of about 12,000 IU/kg BW [
25]. In our case, the vitamin D
3 dose was administered by one shot injection and was approximately 79,000 IU vitamin D
3/kg in crias 1 and 2 and 69,000 IU vitamin D3/kg in cria 3. In blood samples, the vitamin D
3 status is assessed by measuring 25-hydroxycholecalciferol because the formation of 25-hydroxycholecalciferol from vitamin D in the liver is only loosely regulated. The plasma samples of all three examined crias showed severely increased 25-hydroxycholecalciferol concentrations above the upper measuring range (>3750 nmol/L) of the lab. Depending on the reference, the upper reference limits for SACs differ from 200 to 600 nmol/L [
22,
41]. Those upper reference limits for 25-hydroxycholecalciferol are much higher than the reference values from other studies for cattle, sheep, pigs, dogs and cats, which were reviewed by Fairwether et al. [
42]. In humans, vitamin D toxicity occurs due to plasma concentrations of 25-hydroxycholecalciferol consistently above 400 nmol/L. The clinical manifestations result mainly from hypercalcemia and hyperphosphatemia and ectopic tissue calcification when the solubility product of these ions is exceeded [
43].
Crias 1 and 3 received a second shot of vitamin D
3 with a lower dose or as oral paste. Gerspach et al. assumed that such a second dose might be responsible for promoting higher serum vitamin D levels [
22]. Our data do not give any indication of this hypothesis; cria 2 was treated only once with a high dose of vitamin D
3 and also developed severe calcinosis. There might have been some unknown ways of additional oral vitamin D intake, which also have to be considered. In this herd, mineral feed was not supplemented by vitamins, but the mares received a special mash containing 4050 IU/kg vitamin D
3. It cannot be excluded that the crias also ate some of this mash or additional vitamin D
3 was transferred via the colostrum and milk. Different plants, such as
Trisetum flavescens, can also promote calcinosis [
44,
45], but a botanical investigation of the pastures gave no indication of the presence of these plants on the affected farm.
Clinical chemistry of the blood samples of the three alpacas revealed increased creatinine, urea, calcium and potassium. These results are similar to those of the two crias reported by Gerspach et al. [
22] and Jankovsky et al. [
25] and agree with the reports of vitamin D
3 intoxications in adult alpacas we reported earlier [
24]. All crias revealed severe azotaemia; creatinine and urea levels in the plasma of cria 3 were up to 2905 µmol/L for creatinine and 140.3 mmol/L for urea. Other cases of azotemia in SACs were reported after acute kidney failure due to oleander and oak intoxications [
46,
47], or for an alpaca cria with bilateral renal agenesis [
48]. This latter case had highly increased plasma levels of creatinine (1997 µmol/L), urea (46.4 mmol/L) and phosphate (5.8 mmol/L) [
48], whereas the report of another alpaca cria with unilateral renal agenesis indicated only moderately increased levels of creatinine (up to 401 µmol/L) and urea (up to 19.9 mmol/L) [
49]. These discrepancies can be explained by the enormous reserve capacity of the kidney.
However, comparable changes in the kidney do not necessarily have to result from an over-supply of vitamin D
3 in every case. In lambs that also suffered from severe glomerular and tubular kidney damage as a result of nephrosis or toxic tubular necrosis, azotemia (creatinine up to 1273 µmol/L) and proteinuria were also detected by laboratory diagnosis. However, in contrast to the alpaca crias described here, hypocalcemia was present in these animals [
50]. Similar findings were obtained in a fawn in which inflammatory changes in the kidneys were accompanied by azotemia, hyperphosphatemia and also hypocalcemia [
51].
The data of the serum and urine electrophoresis demonstrate that there was a severe renal loss, mainly of albumin, due to insufficient tubular reabsorption.
In cria 1, both plasma and urine proteins were about 40 g/L, which could be explained by a complete destruction of the Bowman’s capsule by the calcification in this animal. In canine urine, an albumin percentage >41.4% and an albumin/alpha-1-globulin ratio >1.46 are an indication of glomerular proteinuria [
52]. The albumin/alpha-1-globulin ratio in cria 1 was 10.17, therefore much higher than the above-mentioned limit for dogs. Smaller proteins (albumins and alpha-1-globulins) were higher in the urine than in the plasma, indicating a dramatic renal loss of those molecules. Additionally, even larger molecules, such as gamma-globulins, were also found in the urine, which reflects complete damage of the glomeruli.
Increasing numbers of destroyed nephrons induced increased urine flow and filtration in the remaining intact glomerula, but tubular reabsorption was probably not increased in an appropriate amount. Subsequently, K+, Na+, water and albumin reabsorption were reduced, resulting in a higher fractional excretion (
Table 5). Particularly in cria 1, a highly increased fractional excretion of potassium was noticeable (
Table 5), which can be explained by the high degree of tubular damage.
The results of the kidney function analysis, as well as the electrophoresis, have to be interpreted with caution. There were no reference values available for kidney function; therefore, the reference values for horses can only serve as an approximation. The reference values for serum proteins from Dawson et al. [
33] had been evaluated for adult animals and not for crias. A previous study investigating serum proteins of camels revealed differences concerning the age of the animals. In serum of adult camels, there was a much higher amount of albumin than in camel crias [
53], which has also to be taken into account when interpreting our results.
In addition to the changes in the kidneys, the necropsy also revealed high-grade changes in other organs, including prominent mineralization in the lungs and liver (
Figure 1,
Figure 2 and
Figure 3). This is consistent with other reports of calcinosis in alpacas or other species where calcifications were found in the lungs and liver [
21,
22,
23,
24]. It can be assumed that mineralization in these organs is further advanced by acute renal failure and that no recovery can be expected from the resulting vicious circle in affected animals. Furthermore, these findings show that for an accurate diagnosis, a detailed medical history should be taken regarding the vitamin supplementation of the animals, since the damage to different organs can lead to a wide range of clinical symptoms. All three animals described here showed similar pathophysiological changes, which were evident both in the values determined by laboratory diagnostics and in the autopsy of the animals. These findings are also consistent with the descriptions of other authors. Nevertheless, it remains questionable why only some of the crias treated with vitamin D
3 (3 out of 12 animals) became clinically ill. This was also observed in the herd described by Jankovsky et al., where previous deaths were attributed to other causes [
25]. Overall, it can be concluded that, on the one hand, the number of cases of undiagnosed vitamin D intoxications in SACs is probably quite high; on the other hand, some animals seem to react more sensitively to high vitamin D doses than others. These results show that more basic research on the vitamin D metabolism in SACs is needed in Central Europe.